Mycobacterium sp. are conventionally identified by microbiological culture. As these microorganisms are slow growing and may exist in low numbers in clinical samples, such culturing techniques are time consuming and may require three to six weeks of culturing before a result is available. Mycobacterium tuberculosis (M.tb) is the causative agent of tuberculosis in humans and is currently diagnosed using these conventional culturing techniques. To prepare for M.tb culture, sputum samples are processed by methods which have been in use for about thirty years (G. Kubica, et al. 1963. Am. Rev. Resp. Dis. 87:775-779). These and other sample processing methods for M.tb culture have been developed for the purposes of 1) reducing the viscosity of the sputum sample to facilitate handling, 2) killing contaminating organisms (such as fungi and non-mycobacteria) to prevent co-culturing with M. tuberculosis and confusion of the diagnosis, and 3) concentrating the sample into a small volume for seeding cultures. To accomplish these goals, conventional sample preparation methods for Mycobacteria sp. have traditionally employed harsh conditions and caustic, toxic or reactive reagents. Conventional sample processing procedures for mycobacterial culture are reviewed by G. Kubica in The Mycobacteria: A Source Book. Part A. (Kubica, G. and Wayne, L. eds.) Marcel Dekker, N.Y. (1984).
A commonly used sample processing method for M.tb culture is the N-acetyl-L-cysteine/sodium hydroxide method. This method uses NaOH, sodium citrate and N-acetyl L-cysteine (NALC) to digest the sample, with recovery of the mycobacteria by centrifugation. The pellet is then resuspended in a small volume and used to inoculate the culture medium. Similar methods have been developed in which sodium hydroxide is used alone, with neutralization of the pellet prior to resuspension and culturing. NaOH has also been used with sodium lauryl sulfate (SLS) to process samples for culture, again with neutralization of the pellet by addition of acid prior to inoculation of the culture medium. The ZEPHIRAN-trisodium phosphate method for sample processing uses trisodium phosphate and ZEPHIRAN (benzalkonium chloride) in a similar process.
Nucleic acid-based genetic methods for identification of microorganisms have greatly reduced the time and labor involved in clinical diagnosis. Such methods include, for example, nucleic acid hybridization (e.g., Southern and slot blots), nucleotide sequencing, nucleic acid cloning techniques, restriction digestion of nucleic acids and nucleic acid amplification. In particular, nucleic acid amplification has provided means for rapid, sensitive and specific identification of microorganisms by amplification and detection of specific genes or gene fragments. However, sample processing for these nucleic acid analyses requires different criteria than sample processing for culturing: 1) nucleic acids must be released from the microorganism in a form suitable for the analysis, 2) nucleic acids must be present in a composition with the appropriate components, ionic strength and pH for the analysis reactions, and 3) inhibitors of the reactions, if present, must be removed. For nucleic acid amplification, certain inhibitors are known to be present in the sample itself, e.g., heine and polysaccharides. In addition, disinfection of the sample is of particular concern for M.tb nucleic acid-based analyses, as such tests are generally not performed in a biosafety cabinet in clinical laboratories.
At the present time nucleic acid-based methods for diagnosis and identification of mycobacteria do not completely replace conventional culturing, as samples which are positive by these methods are generally cultured to determine drug sensitivity. In addition, at the present time the results of nucleic acid analyses are often verified by culture. The need for both conventional culturing and genetic analysis from a single sample has demonstrated that conventional M.tb sample processing for culture is a source of inhibitors which interfere with subsequent nucleic acid-based reactions, particularly amplification. Conventional sample processing for culture is therefore incompatible with many nucleic acid analyses, especially nucleic acid amplification. This incompatibility is believed to be due to the harsh chemical treatment (NaOH, benzalkonium chloride, etc.), which may inhibit the enzymes involved in amplification or render the nucleic acid unamplifiable by other means. Ethylenediamine tetraacetate (EDTA) and lyric enzymes customarily used to release nucleic acids from microorganisms are similarly inhibitory. The conventional phenol/chloroform extraction methods for removing inhibitors may leave traces of these reagents, which are also inhibitory. In addition, residual chaotropes, alcohol or silica, which are conventionally used for purification of nucleic acids may inhibit nucleic acid amplification reactions. The presence of such inhibitors in samples processed by conventional methods has limited the volume of processed sample which can be amplified. That is, to ensure sufficient dilution of inhibitors, only small aliquots (usually less than about 10 .mu.l) of such samples could previously be added to an amplification reaction. Sample volumes above about 10 .mu.l produced erratic results or amplification failures. Even amplification of less than 10 .mu.l of conventionally processed samples have produced erratic results in amplification reactions due to the presence of inhibitors introduced by sample processing. In addition, Applicants have observed that TRITON and other detergents inhibit solid phase assays for detection of amplified DNA.
The ideal sample processing method for nucleic acid amplification of M.tb therefore has the following components: 1) removal of amplification inhibitors, in particular those introduced by sample processing for culture, 2) release from the M.tb of a sufficient amount of DNA for amplification, and 3) disinfection of the sample. Others have attempted to remove amplification inhibitors introduced by conventional M.tb sample processing for culture. Most commonly, these methods involve washing the pellet obtained from sample processing for culture, but sufficient washing to remove inhibitors risks loss of the M.tb organisms with subsequent variability and inaccuracy in culturing results. V. Sritharin and R. Barker (1991. M. Cell. Probes 5:385-395.), A. J. H. Kolk, et al. (1992. J. Clin. Micro. 30:2567-2575.), R. M. Shawar, et al. (1993. J. Clin. Micro. 31:61-65.), P. DelPortillo, et al. (1991. J. Clin. Micro. 29:2163-2168.), and D. V. Cousins, et al. (1992. J. Clin. Micro. 30:255-258.) describe centrifugation washing of a portion of an NALC pellet in specialized lysis buffers containing detergent and/or ethylenediamine tetraacetate (EDTA) and/or lytic enzymes. Shawar, et al. used one washing/centrifugation step. While G. E. Buck, et al. (1992. J. Clin. Micro. 30:1331-1334.) used two microcentrifuge washes of NALC pellets in phosphate-buffered saline, this method was described as having low efficiency and was abandoned. The low efficiency was probably due to inefficient lysis, which was accomplished by either 1 ) 1-12 hr. at 55.degree. C. in a lysis buffer containing enzymes and TRITON, or 2) eight cycles of freezing and thawing. Kolk, et al. also used two washes, but the washes were with lysis buffer which contained detergent and lysis was by exposure to 60.degree. C. for 18 hr. These authors noted failures in their PCR reactions thereafter, and these samples were further treated with phenol/chloroform extraction. T. Victor, et al. (1992. J. Clin. Micro. 30:1514-1517.) described a variation of the washing protocols in which a portion of an NALC pellet was recentrifuged through a sucrose solution. In addition to being time consuming and inconvenient, this method resulted in a 100 fold decrease in sensitivity which would be unacceptable for a diagnostic test.
Other practitioners have attempted to remove amplification inhibitors from samples processed for culture by classical extraction methods using phenol and/or chloroform. These include Cousins, et al. and DelPortillo, et al., supra, as well as P. Shankar, et al. (1991. Lancet 33:5-7.), D. DeWit, et al. (1990. J. Clin. Micro. 28:2437-2441.), C. Pierre, et al. (1991. J. Clin. Micro. 29:712-717.), D. Thierry, et al. (1992. Mol. Cell. Probes 6:181-191.) and A. Brisson-Noel, et al. (1989. Lancet Nov. 4:1069-1071). These methods may not remove all inhibitors and traces of phenol and/or chloroform can contaminate the sample after extraction and inhibit amplification. In addition, these reagents are caustic, flammable and toxic. Commercially-available DNA purification kits (e.g., GENE CLEAN) have also been used to purify M.tb DNA from samples conventionally processed for culture (B. B. Plikaytis, et al. 1991. Mol. Cell. Probes 5:215-219.; K. D. Eisenach, et al. 1991. Am. Rev. Respir. Dis. 144:1160-1163.). This is a time-consuming and cumbersome process which involves many steps and employs a caustic chaotropic binding buffer. Applicants have tested the GENECLEAN method and have found that the sensitivity of subsequent PCR reactions was reduced, most likely because the recovery of M.tb DNA by the GENECLEAN kit was less than recovery using the present method.
Conventional nucleic acid-releasing protocols for mycobacteria have employed enzymatic digestion (Kolk, et al., Cousins, et al., DelPortillo, et al. and Buck, et al., supra), freeze-thaw treatment in detergent-containing buffers (Buck, et al., supra), detergent extraction (DeWit, et al., supra), sonication or sheafing in detergent and/or enzyme containing buffers (Buck, et al. and Savic, et at., supra) or combinations of these techniques (Plikaytis, et al. and Eisenach, et al., supra). As stated above, the detergents, enzymes and EDTA employed in these methods are potential inhibitors of subsequent nucleic acid amplification reactions. Heat has also previously been used to induce lysis of mycobacteria, but again these methods employed lysis in buffers which contained EDTA, detergent and/or lytic enzymes (Sritharin and Barker, Shawar, et al. and Buck, et al., supra) because it was believed that heat alone would not be sufficient to promote efficient lysis. Additional reagents have therefore been customarily used in conjunction with heat to promote efficient lysis.